The acetylation status of core histones plays a pivotal role in regulating gene transcription through the modulation of nucleosomal packaging of DNA (Kouzarides, “Histone acetylases and deacetylases in cell proliferation.” Curr Opin Genet Dev 9: 40-48 (1999); Gray and Ekstrom, “The human histone deacetylase family.” Exp Cell Res 262: 75-83 (2001); Jenuwein and Allis, “Translating the histone code.” Science 293: 1074-1080 (2001)). In a hypoacetylated state, nucleosomes are tightly compacted, resulting in transcriptional repression due to restricted access of transcriptional factors to their targeted DNA. Conversely, histone acetylation leads to relaxed nucleosomal structures, giving rise to a transcriptionally permissive chromatin state. A dynamic balance between the activities of histone acetyltransferases (HATs) and histone deacetylases (HDACs), both of which are recruited to target genes in complexes with sequence-specific transcription activators, maintains this level of this posttranslational modification. Aberrant regulation of this epigenetic marking system has been shown to cause inappropriate gene expression, a key event in the pathogenesis of many forms of cancer (Wade, “Transcriptional control at regulatory checkpoints by histone deacetylases: molecular connections between cancer and chromatin.” Hum Mol Genet. 10: 693-698 (2001); Cress and Seto, “Histone deacetylases, transcriptional control, and cancer.” J Cell Physiol 184: 1-16 (2000); Marks et al., “Histone deacetylases and cancer: causes and therapies.” Nat Rev Cancer 1: 194-202 (2001)). Moreover, evidence demonstrates that inhibition of HDAC triggers growth arrest, differentiation and/or apoptosis in many types of tumor cells by reactivating the transcription of a small number of genes (Jung, “Inhibitors of histone deacetylase as new anticancer agents.” Curr Med Chem 8: 1505-1511 (2001); Grozinger and Schreiber, “Deacetylase enzymes: biological functions and the use of small-molecule inhibitors.” Chem Biol 9: 3-16 (2002); Johnstone, “Histone-deacetylase inhibitors: novel drugs for the treatment of cancer.” Nat Rev Drug Discov 1: 287-299 (2002); Kramer et al., “Histone deacetylase as a therapeutic target.” Trends Endocrinol Metab 12: 294-300 (2001); Marks et al., “Histone deacetylase inhibitors: inducers of differentiation or apoptosis of transformed cells.” J Natl Cancer Inst 92: 1210-1216 (2000)). Xenograft models also confirm these in vitro findings, suggesting that modulation of HDAC's function is a target for the prevention and/or therapeutic intervention of cancer.
To date, several structurally distinct classes of HDAC inhibitors have been reported (Jung, Curr Med Chem 8: 1505-1511 (2001); Grozinger and Schreiber, Chem Biol 9: 3-16 (2002); Johnstone, Nat Rev Drug Discov 1: 287-299 (2002); Kramer et al., Trends Endocrinol Metab 12: 294-300 (2001); Marks et al., J Natl Cancer Inst 92: 1210-1216 (2000)), including short-chain fatty acids (e.g., butyrate, valproate, phenylacetate, and phenylbutyrate) (Lea and Tuisyan, “Discordant effects of butyrate analogues on erythroleukemia cell proliferation, differentiation and histone deacetylase.” Anticancer Res 15: 879-883 (1995); Kruh, “Effects of sodium butyrate, a new pharmacological agent, on cells in culture.” Mol Cell Biochem 42: 65-82 (1982); Newmark and Young, “Butyrate and phenylacetate as differentiating agents: practical problems and opportunities.” J Cell Biochem Suppl 22: 247-253 (1995); Phiel et al., “Histone deacetylase is a direct target of valproic acid, a potent anticonvulsant, mood stabilizer, and teratogen.” J Biol Chem 276: 36734-36741 (2001)), benzamide derivatives (e.g., MS-27-275) (Suzuki et al., “Synthesis and histone deacetylase inhibitory activity of new benzamide derivatives.” J Med Chem 42: 3001-3003 (1999); Saito et al., “A synthetic inhibitor of histone deacetylase, MS-27-275, with marked in vivo antitumor activity against human tumors. Proc Natl Acad Sci USA 96: 4592-4597 (1999)), trichostatin A (TSA) and analogues (Tsuji et al., “A new antifungal antibiotic, trichostatin.” J Antibiot (Tokyo) 29: 1-6 (1976); Jung et al. “Amide analogues of trichostatin A as inhibitors of histone deacetylase and inducers of terminal cell differentiation.” J Med Chem 42: 4669-4679 (1999); Furumai et al. “Potent histone deacetylase inhibitors built from trichostatin A and cyclic tetrapeptide antibiotics including trapoxin.” Proc Natl Acad Sci USA 98: 87-92 (2001)), hybrid polar compounds (e.g., suberoylanilide hydroxamic acid (SAHA)) (Richon et al., “A class of hybrid polar inducers of transformed cell differentiation inhibits histone deacetylases.” Proc Natl Acad Sci USA 95: 3003-3007 (1998); Remiszewski et al., “Inhibitors of human histone deacetylase: synthesis and enzyme and cellular activity of straight chain hydroxamates.” J Med Chem 45: 753-757 (2002)), cyclic tetrapeptides (e.g., apicidin) (Kijima et al., “Trapoxin, an antitumor cyclic tetrapeptide, is an irreversible inhibitor of mammalian histone deacetylase.” J Biol Chem 268: 22429-22435 (1993); Shute et al., “Analogues of the cytostatic and antimitogenic agents chlamydocin and HC-toxin: synthesis and biological activity of chloromethyl ketone and diazomethyl ketone functionalized cyclic tetrapeptides.” J Med Chem 30: 71-78 (1987); Han et al., “Apicidin, a histone deacetylase inhibitor, inhibits proliferation of tumor cells via induction of p21WAF1/Cip1 and gelsolin.” Cancer Res 60: 6068-6074 (2000); Nakajima et al., “FR901228, a potent antitumor antibiotic, is a novel histone deacetylase inhibitor.” Exp Cell Res 241: 126-133 (1998)), and the depsipeptide FR901228 (Nakajima et al., Exp Cell Res 241: 126-133 (1998)). Among these agents, short-chain fatty acids are the least potent inhibitors with IC50 in the mM range, as compared to that of μM or even nM for other types of HDAC inhibitors. Although the use of short-chain fatty acids in cancer treatment has been reported, their therapeutic efficacy has been limited by the low anti-proliferative activity, rapid metabolism, and non-specific mode of action.
Recently, X-ray crystallographic analysis of HDLP (histone deacetylase-like protein), a bacterial HDAC homologue, has suggested a distinctive mode of protein-ligand interactions whereby TSA and SAHA mediate enzyme inhibition (Finnin et al., “Structures of a histone deacetylase homologue bound to the TSA and SAHA inhibitors.” Nature 401: 188-193 (1999)). The HDAC catalytic domain apparently consists of a narrow, tube-like pocket spanning the length equivalent to four to six-carbon straight chains. A Zn2+ cation is positioned near the bottom of this enzyme pocket, which, in cooperation with two His-Asp charge-relay systems, is believed to facilitate the deacetylation catalysis.
Upon careful consideration of other work in the field, we realized that the weak potency of short-chain fatty acids in HDAC inhibition was, in part, attributable to their inability to access the Zn2+ cation in the active-site pocket, which we believe plays a pivotal role in the deacetylation catalysis. Based on this realization and further study, we structurally modified short-chain fatty acids by tethering them with a Zn2+-chelating motif via an aromatic linker. Our discoveries and study have led us to the invention of a new class of Zn2+-chelating motif-tethered short-chain fatty acids, some of which show inhibition of HDAC activity and cancer cell proliferation in nM range, a three-orders-of-magnitude improvement over their parent compounds.